The Role Of Protein Phosphatase 2A In Neuronal Axonogenesis

Protein phosphatase 2A (PP2A) is a principal Ser/Thr phosphatase in the brain. PP2A is indispensable in development, and deficits of PP2A and deterioration of neuronal axons have been observed in several neurodegenerative disorders, but the direct link between PP2A and the neuronal axon development is still missing. Here, we show that PP2A is essential for axon development in transfected rat brain and the dissociated hippocampal neurons. We transfected the EGFP-labeled siPP2Ac or ssiPP2Ac into neuronal progenitor cells of the embryonic rat brains and by in utero electroporation. The growing neurofibers were seen in ssiPP2Ac-injected rats from P3 to P7, whereas largely cells with shorter processes were observed in rats with PP2Ac knockdown by siPP2Ac. At P7, the long axons derived from cortical neurons transfected with siPP2Ac couldn’t acrossed the midline (ML) of the brain. During the establishment of neuronal poalrity, downregulation of PP2A by chemical specific inhibitor (OA), a dominant negative inactive mutant (dnPP2A) or siRNA inhibited axonogenesis, and the axon length, numbers and the percentage of multiple axons decreased significantly.However, upregulation of PP2A by chemical activator (DES) or wtPP2A promotes axon outgrowth and axonogenesis, and the axon number as well as the percentage of multiple axons increased. To explore whether upregulating PP2A could promote axonogenesis in hippocampal neurons with already formed axon, we we treated the neurons with DES or expressed wtPP2A and compared with the vector transfected neurons, the axon length, the numbers and the percent of multiple axons were all increased. To explore whether upregulation of PP2A could rescue the AD-like axon deficits, we treated the primary hippocampal neurons with OA and then transfected into the OA-treated neurons with wtPP2A. Quantitative data showed that compared with OA plus vector group, both axon length and the number were significantly increased in OA plus wtPP2A group. Upregulation of PP2A can promote injury axon regeneration. To explore the possible mechanism involoved in PP2A-induce axongenesis, neurons were transfected with wtPP2A and then immunostained with anti-pT514 antibodies. We found that the phosphorylation level of CRMP-2 at Thr514 decreased both in the cell body and neurites. In N2a cells, expression of wtPP2A resulted in-38% decrement of CRMP2 phosphorylation, whereas knockdown of PP2Ac increased the phosphorylation of CRMP2 at Thr514 to-140% of the control level. Further study showed that both pCRMP2 and tCRMP2 were co-precipitated by anti-PP2Ac, suggesting a direct interaction of PP2Ac with pCRMP2. To further verify the role of CRMP2 dephosphorylation in PP2A-induced axonogenesis, we co-expressed in the hippocampal neurons with wtPP2A and EGFP or Thr514-phosphomimic CRMP2 (T514D-CRMP2), or wtCRMP2, and compared with wtPP2A alone, the axon length, numbers and the percent of multiple axons were all increased by wtPP2A plus wtCRMP2. But the increase was not detected when T514D-CRMP2 was co-expressed with wtPP2A. These data further support that PP2A facilitates axonogenesis by dephosphorylating CRMP2 at Thr514. Lastly, we detected the distribution of PP2Ac in the growing axons at 12 hiv,24 hiv, and 48 hiv respectively. We found that PP2Ac was initially present in all neurites of the unpolarized neurons (12hiv), but it was preferentially enriched in the distal segment of the nascent (24hiv) and maturing axons, extending into the tips of axons (48hiv). These result indicated that PP2A will be required for axonogenesis because of its preferentially localization in the axons. Our results reveal a mechanistic link between PP2A and axonogenesis/axonopathy, suggesting that upregulation of PP2A may be a promising therapeutics for some rieurodegenerative disorders.